Localized Expression of Olfactory Receptor Genes in the Olfactory Organ of Common Minke Whales.

Baleen whales (Mysticeti) possess the necessary anatomical structures and genetic elements for olfaction. Nevertheless, the olfactory receptor gene (OR) repertoire has undergone substantial degeneration in the cetacean lineage following the divergence of the Artiodactyla and Cetacea. The functionality of highly degenerated mysticete ORs within their olfactory epithelium remains unknown. In this study, we extracted total RNA from the nasal mucosae of common minke whales (Balaenoptera acutorostrata) to investigate ORs' localized expression. All three sections of the mucosae examined in the nasal chamber displayed comparable histological structure. However, the posterior portion of the frontoturbinal region exhibited notably high OR expression. Neither the olfactory bulb nor the external skin exhibited the expression of these genes. Although this species possesses four intact non-class-2 ORs, all the ORs expressed in the nasal mucosae belong to class-2, implying the loss of aversion to specific odorants. These anatomical and genomic analyses suggest that ORs are still responsible for olfaction within the nasal region of baleen whales, enabling them to detect desirable scents such as prey and potential mating partners.

Mercury speciation and selenium in toothed-whale muscles.

Mercury accumulates at high levels in marine mammal tissues. However, its speciation is poorly understood. The main goal of this investigation was to establish the relationships among mercury species and selenium (Se) concentrations in toothed-whale muscles at different mercury levels. The concentrations of total mercury (T-Hg), methylmercury (MeHg), inorganic mercury (I-Hg) and Se were determined in the muscles of four toothed-whale species: bottlenose dolphins (n=31), Risso's dolphins (n=30), striped dolphins (n=29), and short-finned pilot whales (n=30). In each species, the MeHg concentration increased with increasing T-Hg concentration, tending to reach a plateau. In contrast, the proportion of MeHg in T-Hg decreased from 90-100% to 20-40%. The levels of T-Hg and Se showed strong positive correlations. Se/I-Hg molar ratios rapidly decreased with the increase of I-Hg and reached almost 1 in all species. These results suggested that the demethylated MeHg immediately formed Se/I-Hg equimolar complex of mercury selenide (HgSe) in their muscles. In addition, an X-ray absorption fine structure analysis (XAFS) of a bottlenose dolphin muscle confirmed that the dominant chemical form of the Se/I-Hg equimolar complex was HgSe. HgSe was mainly localized in cells near the endomysium using electron probe microanalysis (EPMA). These results suggested that the demethylated MeHg finally deposits within muscle cells of bottlenose dolphin as an inert HgSe.

Effects of persistent organochlorine exposure on the liver transcriptome of the common minke whale (Balaenoptera acutorostrata) from the North Pacific.

Hepatic concentrations of persistent organochlorines (OCs) were determined in the common minke whale (Balaenoptera acutorostrata) from the North Pacific. To investigate the effects of OCs on the transcriptome in the minke whale, the present study constructed a hepatic oligo array of this species where 985 unique oligonucleotides were spotted and further analyzed the relationship between the OC levels and gene expression profiles of liver tissues. The stepwise multiple linear regression analysis identified 32 genes that correlated with hepatic OC levels. The mRNA expression levels of seven cytochrome P450 (CYP) genes, CYP1A1, 1A2, 2C78, 2E1, 3A72, 4A35, and 4V6 showed no clear correlations with the concentration of each OC, suggesting that the accumulated OCs in the liver did not reach levels that could alter CYP expression. Among the genes screened by the custom oligo array analysis, hepatic mRNA expression levels of 16 genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. The mRNA levels of vitamin D-binding protein (DBP) were negatively correlated with non-ortho coplanar polychlorinated biphenyl (PCB) levels. Androgen receptor-associated coregulator 70 (ARA70) expression levels showed a significant positive correlation with concentrations of non-ortho coplanar PCB169. These correlations suggest that coplanar PCB-reduced DBP expression could suppress vitamin D receptor-mediated signaling cascades in peripheral tissues. Alternatively, the suppression of vitamin D receptor signaling cascade could be enhanced through competition with the androgen receptor signaling pathway for ARA70. In addition, a negative correlation between kynureninase and PCB169 levels was also observed, which suggest an enhanced accumulation of an endogenous aryl hydrocarbon receptor agonist, kynurenine in the minke whale population. Further studies are necessary to translate the changes in the transcriptome to toxicological outcomes including the disruption of the nervous and immune systems.

Amino acid sequence variations of signaling lymphocyte activation molecule and mortality caused by morbillivirus infection in cetaceans.

Morbillivirus infection is a severe threat to marine mammals. Mass die-offs caused by this infection have repeatedly occurred in bottlenose dolphins (Turiops truncatus) and striped dolphins (Stenella coeruleoalba), both of which belong to the family Delphinidae, but not in other cetaceans. However, it is unknown whether sensitivity to the virus varies among cetacean species. The signaling lymphocyte activation molecule (SLAM) is a receptor on host cells that allows morbillivirus invasion and propagation. Its immunoguloblin variable domain-like (V) region provides an interface for the virus hemagglutinin (H) protein. In this study, variations in the amino acid residues of the V region of 26 cetacean species, covering almost all cetacean genera, were examined. Three-dimensional (3D) models of them were generated in a homology model using the crystal structure of the marmoset SLAM and measles virus H protein complex as a template. The 3D models showed 32 amino acid residues on the interface that possibly bind the morbillivirus. Among the cetacean species studied, variations were found at six of the residues. Bottlenose and striped dolphins have substitutions at five positions (E68G, I74V, R90H, V126I, and Q130H) compared with those of baleen whales. Three residues (at positions 68, 90 and 130) were found to alternate electric charges, possibly causing changes in affinity for the virus. This study shows a new approach based on receptor structure for assessing potential vulnerability to viral infection. This method may be useful for assessing the risk of morbillivirus infection in wildlife.

Effects of temperature on eggs and larvae of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) and its possible role on their geographic distributions.

Effects of temperature on development of eggs, recently hatched larvae and L3 larvae of the marine parasitic nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were examined in vitro. The eggs of A. simplex s.s. hatched at 3-25 °C and those of A. pegreffii hatched at 3-27 °C. Days before hatching varied between 2 days at 25 °C and 35-36 days at 3 °C in A. simplex s.s. and between 2 and 3 days at 27 °C and 65 days at 3 °C in A. pegreffii. Hatching rates of A. simplex s.s. were maintained high at temperatures between 3 and 25 °C but decreased to 0% at 27 °C. In contrast, those of A. pegreffii were lowest particularly at 3 °C, but also at 27 °C. The mean 50% survivals of hatched larvae ranged from 5.3 days at 25 °C to 82.3 days at 9 °C in A. simplex s.s., while in A. pegreffii it ranged from 1.2 days at 27 °C to 77.2 days at 9 °C. L3 larvae of A. pegreffii exhibited higher survival rates and activity than those of A. simplex s.s., particularly at 20 and 25 °C. These results suggest that the early stages of A. simplex s.s. are more adapted to lower temperatures whereas those of A. pegreffii are more tolerant to warm environments, which may correspond to their distribution patterns in Japan and Europe.

Anisakis spp. in toothed and baleen whales from Japanese waters with notes on their potential role as biological tags.

In this study, Anisakis nematodes isolated from toothed and baleen whales from localities around Japan were molecularly (PCR-RFLP) identified. In Wakayama, common bottlenose dolphins (Tursiops truncatus) were infected with A. simplex sensu stricto (s.s.), A. typica and A. pegreffii, while A. typica was the only species found in pantropical spotted dolphin (Stenella attenuata) and striped dolphin (S. coeruleoalba). Offshore common minke whales (Balaenoptera acutorostrata) and sei whales (B. borealis) were almost exclusively infected with A. simplex s.s.. However, in common minke whales from two Hokkaido localities, mature worms mostly consisted of A. simplex s.s. in some individuals and of A. pegreffii in others, but immature worms were mainly A. simplex s.s.. Gross and histopathological examination on gastric mucosa attached by anisakids resulted in mild and superficial reactions by the two baleen whale species in contrast to severe inflammatory reaction associated with ulcer formations by common bottlenose dolphin. Host specificity and adaptability of Anisakis spp. in these baleen and toothed whales were discussed from the points of view of adult worm size, worm population and pathological reactions by hosts. Interestingly, most of the common minke whales predominantly harboring mature A. pegreffii adults belonged to the Yellow Sea - East China Sea stock (J stock), which migrates through the Sea of Japan, whereas most of those mainly parasitized by mature A. simplex s.s. adults were from the Okhotsk Sea - West Pacific stock (O stock), mostly inhabiting the Pacific side, suggesting that these sibling species may have utility as biological tags to differentiate whale stocks. These results represent the first definitive host records for A. pegreffi in the Northwestern Pacific Ocean.

Identification and hepatic expression profiles of cytochrome P450 1-4 isozymes in common minke whales (Balaenoptera acutorostrata).

Full-length cDNA sequences of cytochrome P450 (CYP) 2C78, 2E1, 3A72, 4A35 and 4V6 isozymes were isolated from a hepatic cDNA library of common minke whale (Balaenoptera acutorostrata). The deduced amino acid sequences of minke whale CYP2C78, 2E1, 3A72, 4A35 and 4V6 showed high identities with cattle CYP2C86 (83%), pig CYP2E1 (85%), sheep CYP3A24 (82%), pig CYP4A21 (80%), and human CYP4V2 (76%), respectively. To investigate whether or not these CYP expression levels are altered by contamination of organochlorine contaminants (OCs), mRNA levels of these CYPs in the liver of common minke whale were measured using a quantitative real-time RT-PCR method, and the quantified mRNA levels were employed for the statistical analysis with the residue levels of OCs including PCBs, DDTs (p,p'-DDT, p,p'-DDD and p,p'-DDE), chlordanes (cis-chlordane, trans-chlordane, cis-nonachlor, trans-nonachlor and oxychlordane), HCHs (alpha-, beta- and gamma-isomers) and hexachlorobenzene that have already been reported elsewhere. Spearman's rank correlation analyses showed no significant correlation between CYP expression levels and each OC level in the common minke whale liver, implying that these environmental chemicals have no potential to alter the expression levels of these CYPs or the residue levels encountered in the whale livers may not reach their transcriptional regulation levels. This suggests that the expression of individual CYPs in the whale liver may be at basal level. Relationships among hepatic mRNA expression levels of these CYP2-4 isozymes together with CYP1A1 and CYP1A2 were also examined. Significant positive correlations were detected among mRNA expression levels of individual CYP isozymes in most cases. These associations indicate that the transcriptional regulation of these CYPs examined in this study may be reciprocally related. CYP1A1 levels showed a positive correlation with CYP1A2 levels (r=0.64, p<0.01) indicating that both CYP isozymes were regulated by aryl hydrocarbon receptor activated by endogenous ligands. A strong positive correlation between CYP2C78 and 3A72 (r=0.90, p<0.001) suggests that expression of these CYP isozymes may be under a regulation mechanism of cross-talk in which specific nuclear receptors such as constitutive androstane receptor and pregnane X receptor are involved. The present study indicates that minke whale from the North Pacific may be a model species to investigate the mechanism of basal regulation of these CYPs.

cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins.

We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level.

Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans.

In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994-2010 offshore surveys (21%, 285/1353) and in the 2006-2010 Japanese coastal surveys (20%, 86/436), in Bryde's whales (B. edeni brydei) in the 2000-2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002-2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000-2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000-2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde's whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level.

Molecular cloning and mRNA expression of cytochrome P4501A1 and 1A2 in the liver of common minke whales (Balaenoptera acutorostrata).

This study presents full-length cDNA sequences of CYP1A1 and 1A2, in common minke whale (Balaenoptera acutorostrata) from the North Pacific. Both CYP1A1 and CYP1A2 cDNAs had an open reading frame of 516 amino acid residues, and predicted molecular masses were 58.3 kDa and 58.1 kDa, respectively. The deduced full-length amino acid sequence of CYP1A1 revealed higher identities with those of sheep (86%) and pig (87%), and that of CYP1A2 was most closely related to human (82%) and monkey CYP1A2 (82%) among species from which CYP1A2 has been isolated so far. Differences in certain conserved and functional amino acid residues of CYP1A1 and 1A2 between common minke whale and other mammalian species indicate the possibility of their specific metabolic function. Concentrations of organochlorine compounds (OCs) including PCBs and DDTs analyzed in common minke whale liver showed no significant correlation with hepatic mRNA expression levels of CYP1A1 and CYP1A2, indicating no induction of these enzymes by such OCs.

Spatial and temporal variation of PCBs and organochlorine pesticides in the Antarctic minke whales, Balaenoptera bonaerensis, in the period 1987-2005.

Concentrations of polychlorinated biphenyls (PCBs), dichlorodiphenyl trichloroethane and its metabolites (DDTs), hexachlorocyclohexane isomers (HCHs), hexachlorobenzene (HCB) and chlordane compounds (CHLs) were determined in the blubber of males (20-25 years old) of Antarctic minke whales, Balaenoptera bonaerensis, from the International Whaling Commission (IWC) management Areas IV (70°-130°E) and V (130°E-170°W), south 60°S. The ranges of concentrations (ng g(-1) lipid wt.) for each compound were, PCBs: 7.7-89; DDTs: 29-340; HCHs: 0.20-4.3; HCB: 75-430; CHLs: 10-120, which were much lower than those in common minke whales, Balaenoptera acutorostrata, from the northern hemisphere. The levels of PCBs, HCHs, HCB and CHLs in Area IV were significantly higher than those in Area V, while the levels of DDTs in both areas were similar. For comparing the fate among four pesticides in the Antarctic Ocean avoiding the effect of variance due to food intake, the ratios of the pesticides to PCBs, which has an extremely high chemical stability and environmental persistence, were examined. The HCHs/PCBs ratio decreased by a factor of about 20 in a span of 16 years in both Areas IV and V, while temporal trends of DDTs/PCBs, HCB/PCBs and CHLs/PCBs ratios were not observed. These results indicate that PCBs, DDTs, HCB and CHLs levels did not vary or slightly decreased in Areas IV and V during the study period. However HCHs levels clearly decreased. Spatial differences seems to be related to differences in food intake among whales, and temporal differences seems to be related to the length stay of OCs in the Antarctic Ocean.

Molecular evidence of new variant Brucella in North Pacific common minke whales.

Brucella, a causative agent of brucellosis, has been isolated recently from a variety of marine mammals. The molecular analysis of marine mammalian Brucella strains, without manifest pathology of brucellosis in the eastern North Atlantic, showed that they are distinct from terrestrial Brucella species. Previously, we reported abnormal gonads in common minke whales (Balaenoptera acutorostrata) in the western North Pacific and suggested the presence of Brucella infection in the whales in pathology and serology studies. In the present study, using polymerase chain reaction (PCR), Brucella was detected in granular testes of the whales showing caseation or calcification. The insertion of an IS711 transposable element specific for marine mammal isolates as well as a seal isolate-specific DNA fragment were also found. Molecular characterization of Brucella based on sequence analysis of the PCR products amplified from the outer membrane protein (omp) 2 gene showed that the Brucella from North Pacific common minke whales was different from terrestrial and North Atlantic marine mammal Brucella strains. The North Pacific Brucella showed the highest similarity to North Atlantic seal strains among the known Brucella strains.

Pathological and serological evidence of Brucella-infection in baleen whales (Mysticeti) in the western North Pacific.

Abnormal testes and uterus were observed in 13 males (33%) and one female (3%) out of 40 common minke whales (Balaenoptera acutorostrata) in the western North Pacific. Similar lesions were found in testis and ovary, respectively, in one male (2%) and female (2%) out of 43 Bryde's whales (Balaenoptera edeni) in the western North Pacific. Grossly, granular lesions with caseation and calcification were main pathological signs, and they were restricted to reproductive organs of mature whales. Chronic purulent or granulomatous orchitis was observed by microscopic analysis. Antibodies to Brucella species were detected in the serum samples of 15/40 (38%) of common minke whales and 4/43 (9%) of Bryde's whales. Neither pathological nor serological change was found in the examined sperm whales (Physeter macrocephalus) in the western North Pacific and Antarctic minke whales (Balaenoptera bonaerensis). These results strongly suggest that Brucella infection was involved in two species of baleen whales (Mysticeti) in the North Pacific.

Using trace elements in skin to discriminate the populations of minke whales in southern hemisphere.

Concentrations of 12 trace elements (V, Cr, Mn, Cu, Zn, Se, Rb, Sr, Cd, Cs, Ba, and Hg) were determined in liver and skin tissues of minke whales from various regions within the Antarctic Ocean. Cd concentrations in livers of southern minke whale were apparently higher than those in cetaceans from other regions, while Hg concentrations were lower. There were significant positive correlations between body length and concentrations of Cd and Hg in the liver. The concentrations of all trace elements in the skin were lower than those in other cetaceans reported previously. Significant positive correlations between liver and skin were found for Cr, Mn, Cu, Zn, Rb, Cd, and Cs, implying that the concentrations of these trace elements in the skin reflect those of internal organs. Large interannual variation of the accumulation pattern of trace elements in the skin was observed for the southern minke whales from Area V. There were significant differences in the skin element concentrations among Areas III, IV, and V, especially for males. Also, discriminant analysis between geographically two different groups collected during 1995/1996 austral summer season, based on the concentrations of trace elements in the skin, allowed for a correct classification of 90% of these minke whales. These results suggest that measurement of trace elements in skin samples could provide valuable information on the status of contamination and possible geographic differences in the accumulation levels in southern minke whales.

Searching for novel CYP members using cDNA library from a minke whale liver.

The contaminant-induced cytochrome P450 (CYP) members in minke whale (Balaenoptera acutorostrata) can be potential biomarkers of the contaminant exposure and toxic effects. In this study, we constructed a cDNA library from the liver of minke whale from the North Pacific, and further screened a total of 6930 clones randomly selected in the library for the isolation of cDNA clones encoding novel members of CYP superfamily. The screening revealed the isolation of six novel CYP cDNA clones that are classified into CYP1A, CYP2C, CYP2E, CYP3A, CYP4, and CYP4A subfamilies. The BLAST homology search using the partial cDNA fragments of four CYP subfamilies (CYP1A, CYP2C, CYP2E and CYP4A) demonstrated that the minke whale CYPs were most closely related to pig CYPs (81-91%). Identification of multiple CYP genes in marine mammal species such as minke whale will provide new insights into the metabolic or toxicological functions of individual CYP members.

Production of Sei whale (Balaenoptera borealis) cloned embryos by inter- and intra-species somatic cell nuclear transfer.

The objectives of this study were to choose an effective embryo reconstruction method and an effective post-activation agent for in vitro production of sei whale (Balaenoptera borealis) interspecies somatic cell nuclear transfer (iSCNT) embryos. Moreover, trichostatin A (TSA) treatment of whale iSCNT embryos was performed to improve the in vitro embryo development. In Experiment 1, the fusion rate was significantly higher (88.1%) in embryos reconstructed using the intracytoplasmic cell injection method (ICI) than that (48.7%) in the subzonal cell insertion (SUZI) counterpart. The rates of pseudopronucleus (PPN) formation (77.4 vs. 77.2%) and cleavage (24.5 vs. 37.0%) did not vary between ICI and SUZI. However, the PPN formation and cleavage rates were significantly (P<0.05) lower in the iSCNT embryos than in the parthenogenetic control (95.7% and 64.4%, respectively). Although 21.5% of the bovine parthenogenetic embryos developed to the blastocyst stage, no iSCNT embryo developed beyond the 6-cell stage. In Experiment 2, the cleavage rate did not vary between the TSA (50 nM)-treated and non-treated whale iSCNT embryos (30.5 vs. 32.3%, respectively). Moreover, it did not vary between the TSA-treated iSCNT and SCNT embryos (30.5 vs. 32.0%, respectively). Only one TSA non-treated iSCNT embryo developed to a compacted morula with 20 nuclei. One TSA-treated whale SCNT embryo developed to the 8-cell stage, and out of five whale iSCNT embryos, a 6-cell stage embryo was positive for whale DNA. In conclusion, bovine oocytes have the ability to support development of sei whale nuclei up to the 6-cell stage.

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria.

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel ß-strands and a ß-cup structure consisting of two anti-parallel ß-sheets, respectively. The (TLR4-MD-2)(2) duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.

Validation of high-throughput measurement system with microwave-assisted extraction, fully automated sample preparation device, and gas chromatography-electron capture detector for determination of polychlorinated biphenyls in whale blubber.

Validation of a high-throughput measurement system with microwave-assisted extraction (MAE), fully automated sample preparation device (SPD), and gas chromatography-electron capture detector (GC-ECD) for the determination of polychlorinated biphenyls (PCBs) in minke whale blubber was performed. PCB congeners accounting for > 95% of the total PCBs burden in blubber were efficiently extracted with a small volume (20 mL) of n-hexane using MAE due to simultaneous saponification and extraction. Further, the crude extract obtained by MAE was rapidly purified and automatically substituted to a small volume (1 mL) of toluene using SPD without using concentrators. Furthermore, the concentration of PCBs in the purified and concentrated solution was accurately determined by GC-ECD. Moreover, the result of accuracy test using a certified material (SRM 1588b; Cod liver oil) showed good agreement with the NIST certified concentration values. In addition, the method quantification limit of total-PCB in whale blubbers was 41 ng g(-1). This new measurement system for PCBs takes only four hours. Consequently, it indicated this method is the most suitable for the monitoring and screening of PCBs in the conservation of the marine ecosystem and safe distribution of foods.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes.

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60 approximately 81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Effects of semen extenders and storage temperatures on characteristics of frozen-thawed Bryde's (Balaenoptera edeni) whale spermatozoa.

The present study investigated effects of three semen extenders and storage temperatures on post-thaw characteristics of Bryde's whale spermatozoa. Spermatozoa were collected from the vasa deferens of three mature Bryde's whales captured during the Japanese whale research in the north-west Pacific (May to August 2007) after death. The three semen extenders used for freezing were 1) a commercialized synthetic extender (AndroMed: AM), 2) Tris-based + 10% bovine serum albumin (BSA) and 3) Tris-based + egg yolk (EY). The sperm samples from the three whales were frozen with the three extenders, and the post-thaw spermatozoa were stored at three different temperatures (35 C; 20-25 C, room temperature; and 5 C) for 0, 6, 12, 24, 48 and 96 h. At each time-point, total and progressive motility (PM), viability (live or dead), the hypo-osmotic test, defective acrosomes and malformation were examined. Immediately after thawing, AM resulted in similar recovery rates (60.4 and 83.3%) in 2 of the 3 whales examined and had comparable post-thaw recovery rates to those obtained using the EY and BSA extenders. Immediately after thawing, the proportion of PM in EY (17.6%) was higher (P<0.05) than that in BSA (15.0%). In the hypo-osmotic test, the proportions of AM (26.0%) and BSA (25.2%) were higher (P<0.05) than that of EY (17.3 %). The three extenders had similar viabilities (36.7, 37.9 and 32.1%, respectively), but the viability of BSA was higher (P<0.05) than that of EY. The present study showed that a synthetic semen extender, AndroMed, could be used for cryopreservation of whale spermatozoa in addition to Tris-based extenders containing bovine serum albumin or egg yolk. Storage of the post-thaw Bryde's whale spermatozoa was better at 5 C than at room temperature or 35 C. The frozen-thawed Bryde's whale spermatozoa maintained their motility and viability for at least two days at room temperature and for four days at 5 C.